Generation of Microglia From Human Pluripotent Stem Cells for Neurodegenerative Disease

Modeling
Jeanne Chan1, Erin Knock1, Melanie Kardel1, Adam Anonuevo1, Alym Moosa1, Terry E. Thomas1, Allen C. Eaves1,2, and Sharon A. Louis1
1
STEMCELL Technologies, Vancouver, BC, Canada 2
Terry Fox Laboratory, BC Cancer Agency, Vancouver BC, Canada
H1 (mTeSR" 1) H9 (TeSR™-E8™), IBA1/DAPI
10
5
C D
0.50% 97.95% Percentage of
INTRODUCTION 10
4 Cell Line
CD45+CD11b+
Percentage of TREM2+
3

H1 (mTeSR™1) 95.94% 97.87%

10

CD45
Microglia are critical modulators of neurodegenerative disease. Since microglia are of mesodermal origin, current human 10
2

H9 (mTeSR™1) 98.23% 76.82%

pluripotent stem cell (hPSC)-derived neuroectodermal differentiation models do not give rise to this critical cell type. 10
1

Based on the publications of Abud et al.1 and McQuade et al.2, we have developed media that robustly produce 10
0
0.03% 1.52% H9 (TeSR™-E8™) 98.84% 98.97%
functional microglia-like cells. These cells are distinct from other mononuclear cell types (e.g. monocytes or
0 1 2 3 4 5

H9 (mTeSR™ Plus) 95.91% -

10 10 10 10 10 10
CD11b
macrophages) and express characteristic protein and gene markers across multiple different embryonic stem (ES) and H1 (mTeSR" 1)
STiPS-R038 (mTeSR™1) 90.54% -
H9 (TeSR™-E8™), PU.1/DAPI

induced pluripotent stem (iPS) cell lines maintained on either mTeSR™1, TeSR™-E8™, or mTeSR™ Plus. Additionally, 10
5

96.85% E
WLC-1C (mTeSR™1) 92.03% 93.63%
we show that these cells can be co-cultured with brain organoids and display a reactive-type morphology in response to 10
4

0.00%

injury. 3
STiPS-M001 (mTeSR™1) 92.02% 91.47%

TREM2
10

10
2
STiPS-M001 (mTeSR™ Plus) 90.37% -

METHODS 10
1

0.00%
3.15%
STiPS-F016 (TeSR™-E8™) 98.38% 98.91%
STiPS-B004 (TeSR™-E8™) 98.96% 95.79%
0
10
0 256 512 768 1024
Forward Scatter
Microglia Differentiation: hPSCs maintained in either mTeSR™1, TeSR™-E8™, or mTeSR™ Plus were differentiated into
hematopoietic progenitor cells (HPCs) using STEMdiff™ Hematopoietic Kit (Catalog #05310) for 12 days. On day 12, the FIGURE 3. Optimized Microglia Media Consistently Generate Microglia-Like Cells Expressing Expected Markers by
HPCs were collected and differentiated using the STEMdiff™ microglia culture system for 28 - 34 days (Figure 1). At the Day 34.
end of the maturation stage (day 34), the cells were characterized by flow cytometry. Expression of microglia-specific Representative flow cytometry plots for CD45 and CD11b co-staining (A) and TREM2 (B) versus forward scatter in
genes were assessed by qPCR. IBA1 and PU.1 expression were assessed by immunocytochemistry. Functional microglia generated using the H1 (ES) cell line maintained in mTeSR™1. (C) The percentage of CD45/CD11b
characterization was performed using 1 μg/mL pHrodo™ Red Zymosan Bioparticles™ over a 12-hour incubation co-expression and TREM2 expression from the STEMdiff™ Microglia Culture System across all cell lines tested.
period on an Incucyte® S-3 Live-Cell Analysis System. (D, E) Representative images of microglia stained with IBA1 and PU.1 taken at 20X; scale bars = 50 μm.
H1
H1 H7 H9 STiPS - B004 C
Monocyte Selection and Differentiation: CD14+ monocytes were isolated with EasySep™ Human CD14 Positive H7
STiPS-F016 STiPS-M001 WLS-1C CD14 Fluorescence Intensity in Microglia and Macrophages
Selection Kit II (Catalog #17858) from human peripheral blood leukapheresis packs. hPSCs maintained in mTeSR™1 108
H9

were differentiated using STEMdiff™ Hematopoietic Kit for 7 days. The cultures were switched to STEMdiff™ Monocyte STiPS-B004

CD14 Mean Fluorescence Intensity

10 4
81 10 5

Kit (Catalog #05320). On days 17 - 23, the cells expressed high levels of CD14 fluorescence intensity and were either STiPS-F016

Count
Blood Leukapheresis-isolated Monocytes
54
analyzed by flow cytometry or further differentiated to M1-activated macrophages. STiPS-M001

RNA Expression Levels Relative to

10 3 27
WLS-1C
10 4
M1-Activated Macrophage Differentiation: iPS cell-derived and blood leukapheresis-isolated monocytes were 0
10
0
10
1
10
2
10
3
10
4
10
5
Blood Leukapheresis

differentiated in ImmunoCult™-SF Macrophage Medium (Catalog #10961) for a total of 6 or 8 days. The macrophages 10 2 CD14
10 3
were activated to the M1-phenotype with 10 ng/mL LPS and 50 ng/mL IFN-γ at day 4 or 6 for 2 days before being
collected for flow cytometry.
10 1 H1-derived macrophages
10 2
hPSC-Derived Microglia and Cerebral Organoid Co-Culture: 2.5 - 5 x 105 microglia were added to one well containing H1 microglia

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a single 200-day-old dorsal forebrain patterned brain organoid (BORGs) produced using STEMdiff™ Dorsal Forebrain H9 microglia

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SC ed
10 0

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Organoid Kit for 10 days in STEMdiff™ Neural Organoid Maintenance Medium. An injury was inflicted on the co-culture

er

B
STiPS-B004 microglia

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C
1

9
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-d

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00
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-1
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S1

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9

34
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-d
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Y1

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by piercing the organoid with a 25G needle on day 7.

S-
EM
STiPS-M001 microglia

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hP

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PR

iP
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P2

iP
hP
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TM

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ST
Microglia-Specific Genes WLS-1C microglia
STEMdiff Hematopoietic Kit Protocol Cell Types

Day
-1 0 2 3 5 7 10 12
FIGURE 4. Cells Generated Using STEMdiff™ Microglia Culture System at Day 34 are Distinct From Other
Step Plate iPSC Select wells Harvest supernatant cells
Mononuclear Cell Types.
aggregates with 4 - 10 colonies/cm2 and start STEMdiffTM Microglia
Differentiation Kit protocol
(A) RNA from microglia derived using multiple ES and iPS cell lines shows increased expression of microglia-specific
genes relative to blood CD14+ monocytes (n = 13 - 22). (B) Representative flow cytometry histogram of CD14
Feeding
Full-medium Half- Full-medium Half-medium Half-medium Half-medium
fluorescence intensity for microglia derived from ES (H1 and H9) and iPS (STiPS-B004, STiPS-M001, and WLS-1C) cell
change medium
change
change change change change
lines (unshaded peaks) and H1-derived macrophages (shaded peak). (C) Microglia generated from multiple ES and iPS
Culture Medium cell lines have significantly lower mean intensity of CD14 fluorescence compared to hPSC-derived monocytes
TeSR™
Medium*
Medium A
(Stage 1)
Medium B
(Stage 2) (‘hPSC-derived mono’), hPSC-derived M1-activated macrophages (‘hPSC-derived mac’), and blood M1-activated
*mTeSR™1, TeSR™-E8™, or mTeSR™ Plus macrophages (‘Blood mac’) (n = 2 - 14, p = 0.0002 by two-way ANOVA). The bars show the mean and standard error,
STEMdiff Microglia Culture System
and the dots show the results of individual experiments.

Red Object Count/Max Red Object Count

0 2 12 24 28 - 34
Day

1.0
Step Harvest hematopoietic progenitor cells Collect cell suspension and replate Collect cell suspension and replate Assay ES/iPS cells
microglia M1-activated macrophages
H1 microglia
H9 microglia
Feeding 0.5
STiPS-F016 microglia
Add half-medium volume Add half-medium volume Add half-medium volume
every other day every other day every other day STiPS-B004 microglia
STiPS-M001 microglia
Culture Medium WLS-1C microglia
0.0
STEMdiff™ Microglia STEMdiff™ Microglia 0 1.01.5 7.5 9.5 12.0
Differentiation Medium Maturation Medium
Time (hours)

FIGURE 1. Workflow for hPSC-Derived Hematopoietic Progenitor Differentiation and Microglia Differentiation
and Maturation FIGURE 5. STEMdiff™ Microglia Culture System Generates Functional Microglia Capable of Phagocytosis at Day 34
The amount of pHrodo™ Red Zymosan Bioparticles™ taken up by microglia, blood leukapheresis-isolated M1-activated
macrophages, and ES or iPS cells was measured over a 12-hour time period on an Incucyte® Live-Cell Analysis System.
RESULTS The red object count was divided by the maximum red object count of each experiment.
MAP2/IBA1 MAP2/IBA1

D Percentage of CD43+ Percentage of CD34+/CD45+ Percentage of CD235a+

Cell Line
FIGURE 6. hPSC-Derived Microglia Incorporate into BORGs and Display an Activated Morphology Upon Injury
H1 (mTeSR™1) 95.77 36.16 33.15 (A) Co-cultures with microglia and BORGs stained with IBA1 for microglia (green) and MAP2 for neurons (magenta). The
H7 (mTeSR™1) 97.50 24.53 56.45 microglia integrate among the neurons and display an unactivated morphology with extended processes (arrow).
(B) After a needle-stick injury to the organoid, the microglia display an activated amoeboid morphology as shown by
H9 (mTeSR™1) 97.97 32.30 43.35 IBA1 staining.
WLS-1C (mTeSR™1) 98.18 49.90 12.01

STiPS-M001 (mTeSR™1) - 60.76 58.38

Summary
H1 (TeSR™-E8™) - 48.48 - STEMdiff™ Hematopoietic Progenitor Kit robustly generates HPCs suitable for microglia differentiation.
H9 (TeSR™-E8™) 99.05 39.34 31.03
STEMdiff™ Microglia Culture System robustly generates microglia-like from HPCs across multiple cell lines.
STiPS-F016 (TeSR™-E8™) 98.53 39.05 33.19 These cells:
STiPS-BO04 (TeSR™-E8™) 97.81 40.32 18.84 Express expected microglia markers after 34 days
Have significantly different gene expression patterns and CD14 fluorescent intensity from other
FIGURE 2. STEMdiff™ Hematopoietic Kit Robustly Generates a Starting Population of > 95% CD43+ HPCs by Day 12
mononuclear cells
(A-C) Representative flow cytometry plots for CD43 versus forward scatter (A), CD34 and CD45 co-staining (B), and Are capable of phagocytosis
CD235a versus forward scatter (C) in HPCs generated using the WLS-1C (iPS) cell line maintained in mTeSR™1. (D)
Can be co-cultured with and can integrate into brain organoids for disease modeling
Percentage of CD43- and CD45/CD34-expressing HPCs generated from STEMdiff™ Hematopoietic Kit across all cell
lines tested. References
1. Abud EM et al. (2017) Neuron 94(2): 278–93.e9.
2. McQuade A et al. (2018) Mol Neurodegener 13: 67.

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